Developmental Bias in the Evolution of Phalanges

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Developmental bias in the evolution of phalanges

Kathryn D. Kavanagha, Oren Shovalb, Benjamin B. Winslowa, Uri Alonb, Brian P. Learya, Akinori Kanc,1, and Clifford J. Tabinc,2

aDepartment of Biology, University of Massachusetts Dartmouth, North Dartmouth, MA 02747; bDepartment of Molecular Cell Biology, Weizmann Institute, Rehovat 76100, Israel; and cDepartment of Genetics, Harvard Medical School, Boston, MA 02115

Contributed by Clifford J. Tabin, October 3, 2013 (sent for review April 2, 2013)

Evolutionary theory has long argued that the entrenched rules of invasion of land by amphibians in the Devonian era (8, 9). development constrain the range of variations in a given form, but Clearly, fingers and toes are used for different functions in dif- few empirical examples are known. Here we provide evidence for ferent species, and the number, size, and shape of phalanges a very deeply conserved skeletal module constraining the mor- varies tremendously across tetrapod taxa. Bird toes, for example, phology of the phalanges within a digit. We measured the sizes of allow species to grasp prey, perch on branches, run, paddle, or phalanges within populations of two bird species and found that dig. These different functions correlate with different skeletal successive phalanges within a digit exhibit predictable relative proportions in the series of phalanges bones. Different positions proportions, whether those phalanges are nearly equal in size of the toe joints, which determine proportions, thus are likely to or exhibit a more striking gradient in size from large to small. be selectively advantageous for particular lifestyles (Fig. 1A). Experimental perturbations during early stages of digit formation The extent to which the sizes of the skeletal elements of the demonstrate that the sizes of the phalanges within a digit are limb are truly independent of one another has not been fully regulated as a system rather than individually. However, the sizes explored. Here we address this question from a developmental of the phalanges are independent of the metatarsals. Temporal perspective, focusing on the most distal limb elements, the studies indicate that the relative sizes of the phalanges are es- phalanges. tablished at the time of initial cell condensation. Measurements Results and Discussion of phalanges across species from six major taxonomic lineages showed that the same predictable range of variants is conserved Restriction in the Variation of the Relative Proportions of Phalanges. across vast taxonomic diversity and evolutionary time, starting The phalanges form by a process of sequential segmentation. Cells are added continuously to the distal end of each digit ray with the very origins of tetrapods. Although in general phalangeal “ ” variations fall within a range of nearly equal-sized phalanges to from the so-called digit-forming region (DFR) at the tip (10). When the newly formed cartilage reaches a critical length, a joint those following a steep large-to-small gradient, a novel derived is initiated behind the growing tip, establishing a phalanx behind condition of excessive elongation of the distal-most phalanges has the new joint, and growth of the digit ray continues distal to the evolved convergently in multiple lineages, for example under new joint. The skeletal elements of the limb generally are con- selection for grasping rather than walking or swimming. Even in sidered to have unique individual developmental identities the context of this exception, phalangeal variations observed in specified within the three major limb segments. However, nature are a small subset of potential morphospace. whether this modularity applies to the phalanges has been con- troversial. Some have ascribed a uniquely specified identity to developmental module | developmental constraint | phalanx each phalanx (11), whereas others have suggested that the digits are specified as a whole with the phalanges being generated he impacts of modularity and developmental constraint on through a reiterative segmentation mechanism in the context of Ttrait evolvability are enduring themes in evolutionary developmental biology (1–6). Morphological modules are iden- Significance tified as strongly covarying structures; if the source of this covariance is developmental integration, we expect that de- It has long been proposed that rules stemming from the velopment will produce a particular subset of phenotypic varia- mechanisms used during development can constrain the range tions within populations rather than varying in all possible fl of evolvable variations in a given form, but few empirical directions (4). This biased set of phenotypes then will in uence examples are known. We have focused on developmental evolutionary patterns by limiting options for natural selection. processes determining proportions of phalanx size along in- Theory suggests that evolution of stable integrated develop- dividual digits (fingers/toes) of vertebrates. We find that pha- mentalunitsplaysamajorroleinwhyweseethesetofmor- langeal variation seen in nature is indeed constrained by an phological forms that exist in nature today, in which some ancestral developmental program, limiting morphologies to integrated developmental units become reinforced over gen- a continuum from nearly equal-sized phalanges to a large-to- erations and others break apart or reorganize when such inno- small gradient of relative sizes. Nonetheless, later innovations vations are selectively advantageous (4, 5). As a tool to assess in distal regulation expanded variational possibilities for groups modularity and constraint, morphospace, in which measurements that needed greater grasping ability. These data provide a better are plotted and compared to analyze factors that affect size and understanding of how properties of developmental systems shape, is commonly used as a means to compare differences in work in combination with natural selection to guide evolution morphological form, (7). of skeletal proportions. Modularity in the limb has been widely cited as the basis for the conservation of the basic three-part structure of the limb: the Author contributions: K.D.K., O.S., and C.J.T. designed research; K.D.K., O.S., B.B.W., U.A., stylopod (upper arm or leg), zeugopod (lower arm or leg), and and B.P.L. performed research; A.K. contributed new reagents/analytic tools; K.D.K., O.S., autopod (hand and wrist or foot and ankle). This structural U.A., and C.J.T. analyzed data; and K.D.K., O.S., and C.J.T. wrote the paper. conservation contrasts with the large range of adaptive variations The authors declare no conflict of interest. in size and shape of limb segments among species. How this 1Present address: Orthopedic Surgery, Gifu Central Hospital, Gifu, Gifu 501-1198, Japan. modular structure and variation apply to the most distal limb 2To whom correspondence should be addressed. E-mail: [email protected]. parts, the fingers and toes, has not been explored. Digits evolved edu. much later than the antecedents of the more proximal limb This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. structure and are thought to be neomorphic, arising with the 1073/pnas.1315213110/-/DCSupplemental.

18190–18195 | PNAS | November 5, 2013 | vol. 110 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1315213110 Downloaded by guest on October 1, 2021 However, this previous study determined the timing of the formation of only a single phalanx. To address the timing of phalanx formation more generally, we used two approaches. First, we examined specimens from a fine-scale (2-h) time series through phalangeogenesis in chicks. In addition, we removed one foot of a chicken embryo in ovo, let the embryo grow a variable (4–48) number of hours, harvested the second foot, and then counted the differences in the number of joints formed in the first and second feet to determine the maximum number of hours between the formation of the proximal and distal joints of a given phalanx (SI Appendix). Both sets of observations showed that there is clear variation in the number of hours required to form different phalanges, with the phalanges that ultimately will be the largest having the longest period of formation (∼25 h), and the smaller phalanges having significantly shorter periods (8–15 h) (Fig. 1B). The difference in the timing of formation of the different phalanges could, in principle, reflect distinct rates of the condensation process in different elements; alternatively, the difference could be attributable to distinctions in the size of the Fig. 1. (A) Morphospace potential for bird toe proportions. The x-axis condensations when they first form. To address these possibili- shows the ratio of P2/P1; the y-axis is the ratio of P3/P1. Black rectangles ties, we next measured condensation sizes in 161 chicken em- indicate P1, dark gray rectangles indicate P2, and light gray rectangles in- fi dicate P3. The variations in the toe proportions of a variety of skeletal bryos and 168 Zebra Finch embryos that had been xed during preparations of birds are shown. (B) In chick digits the number of hours the period of phalangeogenesis and then stained with Alcian required for a phalanx to develop from the proximal to the distal joint is Blue cartilage stain and cleared with KOH. The smallest con- related to the size of the initial (distal) condensation. (C) Density of pro- densation size was determined by measuring the area of con- liferating cells (BrdU-incorporated cells) is relatively high in the distal- densation just after the proximal joint of the phalanx was forming tip of digits, but once the distal joint is formed on a phalanx, pro- initiated. Down-regulation of cartilage matrix precedes forma- liferation is reduced. No significant differences in proliferation rate are tion of the joint interzone (i.e., the Alcian Blue staining becomes found among formed phalanges within a digit. (D) The size of the initial clear), and thus the earliest stages of joint position can be seen fi condensation is correlated with size of the condensation when the nal easily in Alcian Blue-stained hind limbs. In evaluating whether pattern is achieved (day 10) in digits III and IV in chicken embryos. our sample size was sufficient to detect the initial condensations, we considered that five samples within 5% of the smallest measurement indicated a sampling level at which the smallest the digit identity (12). To differentiate between these hypothe- fi ses, we examined the variation in phalanx size within a single initial condensation size was known with con dence (SI Appen- fi dix). We found that initial condensation sizes differed among species. We reasoned that if phalanges are speci ed in- fi fi dependently of one another, then the sizes of different homol- phalanges and were signi cantly correlated with nal phalanx < ogous phalanges should vary independently among individuals. In size (P 0.0001, ANOVA) (Fig. 1D). contrast, if phalanges are established as part of a developmental module, then their proportions should covary. We measured the 2D area of the first, second, and third phalanges (P1–P3) of the fourth hind limb digit (digit IV) from radiographs of large col- lections of adult chicken and Zebra Finch skeletons. For each individual we plotted the ratio of P3/P1 versus the ratio of P2/P1, EVOLUTION with each individual represented by a single point in morphospace. Strikingly, the plotted ratios for both species fall closely along asingleline(Fig.2A). With knowledge of this relationship, one can predict the size of P3 accurately by knowing the sizes of P1 and P2. To verify that the apparent relationship between phalanges reflects a meaningful developmental integration in the formation of successive digits, we conducted simulations in which phalanx sizes were reassigned in series. The results substantiate that sizes of phalanges are highly unlikely to vary independently (P < 0.001) (SI Appendix).

Time and Size of Formation of Individual Phalanges. The lack of independence among phalanges suggests that there might be Fig. 2. Variation in proportions of phalanges within and among vertebrate a developmental linkage between the formation of successive groups. All groups vary along a line from equal-sized to a proximodistal phalanges in a growing digit. Previous studies have noted that gradient. Ratios are taken from area measurements and are standardized against the size of the ﬁrst phalanx. x-axis: P2/P1; y-axis: P3/P1. Plots of the there is a cyclic oscillation in the expression of the Hairy2 gene in – the developing chick autopod, resembling the activity of the gene proportions of individual phalanges are for digit IV, P1 3in(A) individual Zebra Finch and chick populations, (B)birds,(D) lissamphibians, and reptiles. In segmentation clock during somitogenesis (13). Moreover it was hyperphalangic (C) early sarcopterygians, (F)cetaceans,(E) ichthyosaurs, and noted that the period of this oscillation, 6 h, is precisely half that plesiosaurs, the plots are ratios of multiple sets of three sequential phalanges. of the formation of a phalanx, in particular the second phalanx Equations for the lines are Zebra Finch, y = 0.7528 + 0.1412, r2 = 0.43; chick, y = (P2) of wing digit II, suggesting that phalanges could be gener- 0.63 × 0.03, r2 = 0.49; P < 0.0001; birds, y = 1.147 × −0.19, r2 = 0.77; early ated on a periodic basis every two cycles of a clock (13). If true, sarcopterygians, y = 0.944 × −0.17, r2 = 0.62; lissamphibians, y = 0.743 × −0.03, this observation might suggest that segments would form at r2 = 0.605; reptiles, y = 0.949 × −0.08, r2 = 0.723; cetaceans, y = 1.21 × −0.39, a constant rate and on a constant scale, with differences in r2 = 0.825; ichthyosaurs, y = 1.23 × −0.32; r2 = 0.71; plesiosaurs, y = 1.327 × phalanx size being attributable to subsequent differential growth. −0.44; r2 = 0.854.

Kavanagh et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18191 Downloaded by guest on October 1, 2021 Although different phalanges form on a scale proportional to not disruptive of phalanx length. Importantly, the impermeable their final size, differences in the lengths also could be influenced barriers not only affected the next forming phalanx but also led by differential rates of growth. Therefore, we next used BrdU to subsequent phalanges being of extended length, even though incorporation to evaluate the proliferation rates of the newly they were formed at a distance from the barrier and well after formed phalanges to evaluate the alternative hypothesis, i.e., that the digit had healed from the manipulation (Fig. 3). In essence, differing proliferation rates determine differing phalanx sizes. after insertion of the barrier, the still-to-be-formed phalanges Although there is a higher rate of proliferation in the distal tip, behaved as a modular set, seemingly starting a decreasing se- where the DFR contributes cells to the condensation distal to the quence anew, distal to the barrier. In most cases the overall most recently formed joint, we saw no significant differences in length of the digit with the barrier insertion was very close to that the percentage of proliferating cells within the different pha- seen on the contralateral side, indicating that only the modular langes (i.e., segments that have both proximal and distal joints; segmentation process, and not the overall growth parameters, Fig. 1C). Thus, although proliferation may amplify differences in had been affected. In addition, as expected, the terminal pha- the relative size of the phalanges (larger phalanges having more lanx, which is known to be formed by a process different from total proliferating cells), the essential differences in relative that forming the proximal phalanges (15), always was unaffected. phalanx size appear to be determined by the length of time that Many influential evolutionary studies treat the metapodials as has elapsed and hence the number of additional cells added to separate from the phalanges, likely because they are observed to the growing segment before a new joint forms. vary independently of the loss of the digits (16–18). In contrast to their obvious functional and variational independence from Testing Developmental Modularity Within a Digit. Within a given the phalanges within a digit, the morphogenesis of metapodials digit, the high degree of correlation in the variation of phalanx appears to be exactly the same as that of the phalanges. Thus, the size suggested that phalanges are likely to develop as a modular metacarpal and metatarsal bones often are viewed as forming unit. However, the different phalanges within a module are not from the same “digital rays” as the phalanges distal to them. No all the same size but rather decrease successively in size. A histological or structural differences or differences in gene ex- parsimonious explanation would be that each phalanx negatively pression have been reported (19–21). Here, we used an experi- fl in uences the size of the next, to an extent characteristic of each mental approach to test whether metatarsals are part of the same digit. Because we have found that the proportions of the various developmental module as the phalanges. We inserted foil bar- phalanges are established at the time of prechondrogenic con- riers in joint-forming regions between the metatarsal and the densation, such a negative influence, if it exists, must act at the fi presumptive proximal phalanx on day 5.5 of chick development, time when the cartilage anlagen is rst being set aside (14). To and embryos were fixed at day 10 when the tip had formed. We test this notion, we inserted a thin foil barrier into the distal found that, although the metatarsals were consistently and var- phalanx-forming region, sometimes ending up in the forming fi iably shortened (25/25 of affected digits were shortened), the joint interzone between the rst and second phalanx at day 6.5 in phalanges developed normally 100% of the time (Fig. 3). Thus, the chick. The hope was that such a barrier would effectively fl fl the metatarsals do not appear to in uence the segmentation block the in uence of the proximal differentiating phalanx on process forming the phalanges and accordingly appear to be a the formation of the next. Indeed, such manipulations consis- distinct developmental module. tently resulted in the next forming phalanx (P2) being significantly longer than seen in the unoperated contralateral digit The Impact of Modular Development on the Evolution of Phalanges. (n = 28/36) (Fig. 3). This phenotypic effect was rarely seen in = We have found that the phalanges of a given digit are formed as wound controls (n 2/27), indicating that the operation itself was a module. Therefore, although the absolute sizes of homologous phalanges vary within a population, the set of phalanges in any digit maintains predictable size ratios. One consequence of this limit on the range of variation within a population would be a limited range of phenotypes upon which selective forces can act. Thus, in theory, the modular development of the phalanges within a digit would be expected to result in a consequent bias in the evolution of phalangeal patterns. To look at this possibility, we examined the ratios of phalangeal sizes within representative individuals from all available vertebrate taxa that have three or more phalanges in a series (excluding the clawed tip). Area (2D) measurements were taken from digital images of articulated skeletons or fossils or from radiographs of museum specimens. Size proportions were plotted separately for each major group. In some species the sequential phalanges in a given digit are almost the same size, whereas in others there is a dramatic decrease in size from phalanx to phalanx. However, the ratios of the sizes of the phalanges within these digits form a set of proportion variations that is consistent in all taxa (Fig. 2). For example, in birds, when proportions are plotted with P2/P1 vs. P3/P1, the variational distribution of proportions of digit IV, P1–3in all birds appears to be an extension of the variation found among individuals in chick and Zebra Finch populations (Fig. 2). Sta- tistical analysis shows a high likelihood that both chick and Ze- bra Finch populations fit the same regression line, which is not detectably different from the regression line for all bird species (P < 0.0001) (SI Appendix). These results demonstrate that the influence of modular development on variation within bird Fig. 3. Barrier experiments. Tantalum foil barriers inserted into developing fl phalanges affect the size of the distal phalanges. Barriers inserted into de- populations has indeed in uenced evolution within class Aves as veloping metatarsals affect metatarsal length but not the patterning of the a whole. The same restriction in phalangeal variation is seen in phalanges. The top digit in each pair is the contralateral control. Proximal is other taxonomic groups, including land reptiles, plesiosaurs, to the left. Images are aligned by metatarsal–phalangeal joint (MTPJ). ichthyosaurs, amphibians, and cetaceans. The variation is limited

18192 | www.pnas.org/cgi/doi/10.1073/pnas.1315213110 Kavanagh et al. Downloaded by guest on October 1, 2021 so that, by knowing the sizes of two phalanges, one can reliably predict the size of a third. To see if the developmental integration in the formation of the digital skeleton and the consequent predictability of relative phalanx sizes is a basal feature of tetrapods, we measured the phalanges present in the earliest autopods known from the fossil record, including several extinct amphibian species (22, 23), and from lungﬁsh embryos, because they are a living representative of a basal sarcopterygian, the group from which tetrapod vertebrates evolved (24). The ratios of phalanx sizes in these early autopods are similar to those seen in modern tetrapods, suggesting that the digit skeletal elements have been formed as a developmental module with biased variations since the origina- tion of the autopod, with metapodials later evolving into a separate module. Indeed when each major modern taxon was plotted against basal groups as a whole, no signiﬁcant differences in slope were observed (Fig. 2 and SI Appendix).

Innovation in the Evolution of the Distal Phalanges in Derived Groups. There is one major and very informative exception to this overall pattern. In the hindlimbs of some birds and in the forelimbs of some bipedal dinosaurs and climbing or digging reptiles, the penultimate phalanx (the last phalanx before the tip) is elongated. All these exceptions apparently are coincident with the lack of primary dependence on the limb for propulsion. For example, after examining digital images of a diverse sample of 76 species of birds, P4 of digit IV (and sometimes P3 of digit III) was seen to be significantly longer than anticipated, based on the foregoing analysis, in a number of different species (Fig. 4 and SI Appendix). Differences in the relative length of the penultimate phalanx of digits III and IV has been noted previously as a key variable that has been interpreted functionally relative to the lifestyle of birds along a simple spectrum from arboreal to cur- sorial (25, 26). However, these prior studies did not consider the penultimate phalanges in the context of the overall proportions of the proximal phalanges. To gain a better understanding of the variation in digit IV, we plotted the data for digit IV in 3D morphospace (plotting the Fig. 5. (Top) Phalanges proportions for digit IV in birds fall roughly within ratios of P2/P1, P3/P1, and P4/P1, along the three Cartesian a triangular plane in 3D morphospace. (Middle) Chick (white dots) and Zebra axes) (Fig. 5 and SI Appendix). If digit IV, P4 were regulated Finch (black dots) individuals fall within the same morphospace plane as fi independently, all points would be expected to be directly ver- proportion variations among species. (Bottom) Galapagos nch species that – are more arboreal (yellow) have altered proportions as compared with more tical in the z plane above the x y line. In contrast, we found the + observed proportions fell within a roughly triangular plane that ground-dwelling species (black). Ellipses are centroid 1SD. was significantly off the vertical. This “tilted” position of the

plane results from the observation that, in the species with the EVOLUTION by the basal pattern (see screech owl digit IV in Fig. 4). One most elongated P4s, the P3 is also slightly larger than expected plausible developmental model for this pattern of distal elongation would be a response to a distal signal extending growth and delaying segmentation of the distal phalanges superimposed on the general developmental program leading to proportional decrease in sequential phalanx size. As is consistent with this model, the phalangeal elongation we observe is always in the distal-most phalanges. This pattern also is reﬂected in the triangular shape of the plane, which indicates that some con- ceivable variations in proportion on the plane are never found in nature (e.g., long-short-long-short). This restricted morphospace is highly improbable unless all phalanges covary as part of the same module (SI Appendix) (27). In other words, compared with the majority of vertebrate digits, the distal-most phalanges in birds can become relatively longer than expected from the basal pattern, creating an increased range of variability for these groups. Variation among individuals within populations also falls within this same morphospace (Fig. 5B). We next tested whether particular proportions in birds were correlated with function. Birds were categorized according to Fig. 4. Examples of convergence in phalanges proportions. From left to known lifestyles or behaviors involving toes of the particular spe- right, P1–P4 are aligned; ungual phalanges (tips) have been removed. Pha- langes proportions evolve along predicted lines, e.g., the Ground Hornbill, cies. The functional categories included raptors, perchers, diggers, a walking species whose closest taxon is primarily perching, has evolved walkers, swimmers, and generalists, with species categorized based toward the walking variant by elongating P1 and developing a steeper on published literature. We found that particular proportions in proximodistal gradient. 3D space were associated with particular functional categories

Kavanagh et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18193 Downloaded by guest on October 1, 2021 is reduced for a given limb, the distal phalanges appear to be able to elongate dramatically, particularly in association with perching and grasping functions. Moreover, this evolution can occur relatively rapidly. We examined the range of variation in digit IV, P4 in a group of species that have undergone a relatively recent adaptive radiation, the Darwin’s finches, which evolved in the Galapagos Islands within the last 3 million years (30). We found that in the more arboreal finches the distal phalanges are more elongated, consistent with the overall correlation between arbor- eality and P4 length among all birds. The variation in these finches again remains within the predicted plane in 3D morphospace (Fig. 5C). Birds that are diggers, perchers, and raptors have eluded the constrained range of variation in phalangeal proportions through a developmental innovation that apparently has been layered on top of the basal modular phalanx program, allowing greater range of variation in the size of the distal phalanx than other- wise possible. In arboreal and digging mammals, e.g., in Grant’s Golden Mole (31) and in certain extinct taxa of sloths (32), this evolutionary challenge has been circumvented differently, by condensing phalangeal elements in normal proportions but then fusing phalanges with one another or with the metapodial to produce a long segment from shorter phalanges. Other arboreal Fig. 6. Ellipses showing the patterns of the phalanges by functional groups mammals, such as the Two-Toed Sloth (33, 34), have paralleled established by known lifestyles of the birds. Ellipses are defined by the cen- the digging/perching/predatory birds in extending their distal troid + 1 SD in all directions for each functional group. phalanges to a greater extent than the proximal ones. An insight into at least a potential mechanism by which this extension might have occurred comes from a study (15) in which Fgf signaling (Fig. 6), with evolution occurring along lines in morphospace that was maintained experimentally at the distal tip of a growing digit suggest tradeoffs between competing functions (27). ray. The result was an elongation of the penultimate phalanx, Indeed, by comparing variations found within and between although the terminal claw was of normal proportions (as is the related groups, we found evidence of convergence for function case in birds with a naturally extended penultimate P4 of digit in several groups. For example, the secretary bird (Sagittarius IV). Thus, one might postulate that, in addition to the modular serpentarius) is from a raptor clade, but its toes function not only developmental program influencing the developing phalanges, for grasping prey on the ground but also for walking (28, 29). The the length of the phalanges also is positively influenced by signals proportions of the secretary bird’s phalanges are intermediate such as Fgf produced at the tip of the DFR. One also could between those of raptors and flightless birds (walkers), with a P1 postulate that maintaining or increasing the level of this distal that is greatly elongated compared with that of any other raptor signal can superimpose an extension of the distal-most phalanges we measured (Fig. 4). In other words, given sufficient time and on the normal pattern of regularly decreasing phalanx size. selective pressure, the proportions of phalanges in bird digits evolve as a module along predictable lines in morphospace (27), Conclusions e.g., between optimized walking and grasping functions (Fig. 6). Although previous studies have reported that proportions of Thus, despite the highly conserved relationships among pha- limbs and other skeletal elements are evolvable but are signifi- langeal sizes attained in evolution, when dependence on walking cantly constrained within and between segments (18, 35–37), the

Fig. 7. Summary of results. (A) Variants in phalanx proportions remain within a predictable range, from a large-to-small gradient in walkers to equal-sized in perchers to distal elongation of the penultimate phalanx in raptors. Walking and perching specialist phenotypes (W, P) are connected in morphospace by a line, where different phenotypes along the line show a trade off of functions (27). Three functions lead to a full triangle whose vertices are W, P, and the raptor specialist phenotype, R. Joint positions are determined in sequence as the digit grows. Distal elongation may be caused by the in- hibition of the joint-initiation signals. (B) Hypothesis of origins of modules in the digit. Phalanges in early tetrapods vary as one module, whereas derived tetrapods have two independently varying modules. MT, metatarsal; ph, phalanx.

18194 | www.pnas.org/cgi/doi/10.1073/pnas.1315213110 Kavanagh et al. Downloaded by guest on October 1, 2021 developmental basis for these observations has been unclear. analysis data were used to measure variance and to establish the rela- Focusing on the distal elements of the autopod, we have found tionships between various sets of proportions. A statistical protocol was that the formation of the phalanges of each digit is specified as used on generated randomized datasets to establish whether the ob- a developmental module rather than as a series of independently served relationships between phalanges proportions were statistically sig- specified elements. As a consequence, there is a developmental nificantly different from random (described in detail in SI Appendix). Functional bias, so that in independent vertebrate lineages the proportions of data on the use of toes by bird species were obtained from published sources phalanges have evolved repeatedly within a limited subgroup of all (SI Appendix). possible variations in morphospace (Fig. 7). The variation arises developmentally during a short period of morphogenesis during Developmental Analyses. We used two methods to calculate the number of which joint positioning is established. The scenario we propose hours elapsed between the formation of the proximal joint and the for- involves an ancestral developmental system that allowed variations mation of the distal joint in a phalanx. First, several large batches of chick ranging from equal-sized to a large-to-small gradient, with later eggs were incubated synchronously, and a 2-h time series was collected and innovations in distal regulation that opened up new variational stained with Alcian Blue. Condensation sizes for each phalanx were measured possibilities for groups that needed greater grasping ability. These over each time period. The average elapsed time between proximal and distal data thus provide a better understanding of how the properties of joint formation was calculated to estimate the time of formation for a given phalanx. A second method involved removing one limb, allowing the embryo developmental systems work in combination with natural selection – to guide evolution of skeletal proportions in vertebrates. to grow for 4 48 h longer, then removing the other limb, performing Alcian staining, and counting the difference in the number of joints. The maximum Methods number of hours before a new joint was observed in a digit was determined by estimating the number of hours required to form a given phalanx. Pro- Analysis of Proportions and Morphospace. Adult specimens of birds, reptiles, liferation rates were calculated by labeling developing embryos with BrdU amphibians, cetaceans, plesiosaurs, ichthyosaurs, and Darwin’s finches were (Invitrogen EdU kits Alexa Fluor 555) and counting the numbers of labeled obtained from the American Museum of Natural History, the British Museum cells in 200-μm quadrants. Barrier experiments were conducted using tan- of Natural History, the Harvard Museum of Comparative Zoology, the Bos- talum foil implants into digit IV of chick hindlimbs either on day 5 (meta- ton Museum of Science, and the Museum of the Rockies (see Dataset S1). tarsal barriers) or on day 6–7 (phalanges barriers). Embryos were collected at Zebra Finch feet were obtained from university research animal colonies. day 10, fixed, and Alcian stained. For wound controls foil barriers were The birds were excess breeders that were part of IACUC approved protocol BU11-026 in an AAALAC approved songbird breeding facility at Boston inserted and then were removed about 1 min later. Alcian Blue-stained University; Chicken feet were obtained from Boston Chinese markets. From condensations were measured and compared among treatment groups. skeletal preparations and fossils, digital photographs were taken from the dorsal (top) side of the foot; for museum skins, digital X-ray was used to ACKNOWLEDGMENTS. We thank the members of the C.J.T. and K.D.K. lab- obtain images of phalanges. For some of the early sarcopterygian taxa, oratories for comments on the research and manuscript, L. Mahler and S. Mallick for assistance with data analysis, C. Koeppl for providing embryos, which are extremely rare and difficult to access, published photographs or and the British Museum of Natural History and Museum of Comparative illustrations were used to obtain measurements. Measurements of phalan- Zoology at Harvard University for access to specimens. This work was sup- ges areas were made using ImageJ software by tracing around each phalanx. ported by National Institutes of Health/National Institute of Diabetes and Proportions (P2/P1, P3/P1, and P4/P1) were plotted in 2D or 3D morphospace Digestive and Kidney Diseases Grant P01DK056246 (to C.J.T.) and funds from for visualization of the variation. Using MatLab, principal component the University of Massachusetts Dartmouth (to K.D.K.).

1. Atchley WR, Hall BK (1991) A model for development and evolution of complex 21. Storm EE, Kingsley DM (1999) GDF5 coordinates bone and joint formation during morphological structures. Biol Rev Camb Philos Soc 66(2):101–157. digit development. Dev Biol 209(1):11–27. 2. Gould SJ (1977) Ontogeny and Phylogeny (Harvard Univ Press, Cambridge, MA). 22. Coates MI, Clack JA (1990) Polydactyly in the earliest known tetrapod limbs. Nature 3. Kirschner M, Gerhart J (1998) Evolvability. Proc Natl Acad Sci USA 95(15):8420–8427. 347:66–69. 4. Klingenberg CP (2008) Morphological integration and developmental modularity. 23. Carroll R (2009) The Rise of Amphibians: 365 Million Years of Evolution (Johns Hop- – Annu Rev Ecol Evol Syst 39:115 132. kins Univ Press, Baltimore). 5. Schlosser G, Wagner G (2004) Modularity in Development and Evolution (Univ of 24. Ahlberg PE 2003. Fossils, developmental patterning, and the origin of tetrapods. The Chicago Press, Chicago). New Panorama of Animal Evolution, eds Legakis A, Sfenthourakis S, Polymeni R, 6. West Eberhard MJ (2003) Developmental Plasticity and Evolution (Oxford Univ Press, Thessalou-Legaki M (Pensoft Publishers, Moscow) pp 44–54. New York). EVOLUTION 25. Hopson JA 2001. Ecomorphology of avian and non-avian theropod phalangeal pro- 7. Eble GJ (1999) Developmental and non-developmental morphospaces in evolutionary portions: Implications for the arboreal vs terrestrial origin of birds. New Perspectives biology. Morphospace Concepts and Applications, eds Chapman RE, Rasskin-Gutman D, Wills M (Cambridge Univ Press, Cambridge, UK). on the Origin and Early Evolution of Birds: Proceedings of the International Sympo- 8. Hall BK (2007) Fins into Limbs: Evolution, Development, Transformation (Univ of sium in Honor of John H. Ostrom, eds, Gautheir J, Gall LF (Peabody Museum of Chicago Press, Chicago). Natural History, Yale University, New Haven, CT). 9. Shubin N, Tabin C, Carroll S (1997) Fossils, genes and the evolution of animal limbs. 26. Kambic RE 2008. Multivariate Analysis of Avian and Non-Avian Theropod Pedal Nature 388(6643):639–648. Phalanges. M.S. thesis (Montana State University, Bozeman, MT). 10. Suzuki T, Hasso SM, Fallon JF (2008) Unique SMAD1/5/8 activity at the phalanx- 27. Shoval O, et al. (2012) Evolutionary trade-offs, Pareto optimality, and the geometry of forming region determines digit identity. Proc Natl Acad Sci USA 105(11):4185–4190. phenotype space. Science 336(6085):1157–1160. 11. Wolpert L (2002) Limb patterning: Reports of model’s death exaggerated. Current 28. Steyn P (1983) Birds of Prey of Southern Africa: Their Identification and Life Histories Biology 12(R):R628–R630. (Tanager Books, Inc., Dover, NH). 12. Richardson MK, Jeffery JE, Tabin CJ (2004) Proximodistal patterning of the limb: In- 29. Ferguson-Lees J Christie. D. 2001. Raptors of the World. (Houghton–Mifflin Company, – sights from evolutionary morphology. Evol Dev 6(1):1 5. New York). 13. Pascoal S, et al. (2007) A molecular clock operates during chick autopod proximal- 30. Grant BR, Grant PR (2002) Lack of premating isolation at the base of a phylogenetic – distal outgrowth. J Mol Biol 368(2):303 309. tree. Am Nat 160(1):1–19. 14. Hartmann C, Tabin C (2001) Wnt-14 plays a pivotal role in inducing synovial joint 31. Kindahl M (1949) The embryonic development of the hand and foot of Eremitalpa formation in the developing appendicular skeleton. Cell 104(3):341–351. (Chrysochloris) grani (broom). Acta Zool 30:133–152. 15. Sanz-Ezquerro JJ, Tickle C (2003) Fgf signaling controls the number of phalanges and 32. Naish D (2005) Fossils explained 51(6): Sloths. Geology Today 21:232–238. tip formation in developing digits. Curr Biol 13(20):1830–1836. 33. Mendel FC (1981a) Foot of two-toed sloths: Its anatomy and potential uses relative to 16. Gatesy SM, Middleton KM (1997) Bipedalism, flight and the evolution of theropod size of support. J Morphol 170:357–372. diversity. J Vertebr Paleontol 17:308–329. 34. Mendel FC (1981b) Use of hands and feet of two-toed sloths (Choloepus hoffmanni) 17. Shapiro M, Hanken J, Rosenthal N (2003) Developmental basis of digit loss in the during climbing and terrestrial locomotion. J Mammal 62:413–421. Australian lizard Hemiurgis. J Exp Zool B Mol Dev Evol 297:48–56. 35. Adams DC, Nistri A (2010) Onotogenetic convergence and evolution of foot mor- 18. Young NM, Wagner GP, Hallgrímsson B (2010) Development and the evolvability of human limbs. Proc Natl Acad Sci USA 107(8):3400–3405. phology in European cave salamanders (Family: Plethodontidae). BMC Evol Biol Jul 19. Guo X, et al. (2004) Wnt/beta-catenin signaling is sufficient and necessary for synovial 16:10:216. joint formation. Genes Dev 18(19):2404–2417. 36. Kavanagh KD, Evans AR, Jernvall J (2007) Predicting evolutionary patterns of mam- 20. Settle SH, Jr., et al. (2003) Multiple joint and skeletal patterning defects caused by malian teeth from development. Nature 449:427–432. single and double mutations in the mouse Gdf6 and Gdf5 genes. Dev Biol 254(1): 37. Young NM (2013) Macroevolutionary diversity of amniote limb proportions predicted 116–130. by developmental interactions. J Exp Zool B Mol Dev Evol 320(7):420–427.

Kavanagh et al. PNAS | November 5, 2013 | vol. 110 | no. 45 | 18195 Downloaded by guest on October 1, 2021